In bio-reactor technology large-scale production of bio-active compounds, including bio-polymers, from bacterial micro organisms is currently performed in batch fermenters and requires product purification downstream. One of the major limitations of the batch fermentation technology is the large volume of the fermenter needed to accomplish this task on a mass-scale (in the order of millions of tons/year) and the high cost for installing and operating such fermenters.
As an example, most recombinant antibodies approved for human use are currently produced by large-scale cultivation of mammalian micro organisms (e.g. Chinese Hamster Ovary cells, or CHO cells). The process is expensive because of its limited capacity. The investment needed to establish a culture unit for mammalian cells is substantial (approximately 10 million Euro is required for an antibody production of 1 kg/year).
By 2002 approximately 64 different Monoclonal Antibodies (MAbs) had been approved by FDA (US Food and Drug Administration)—a figure expected to rise to 500 in less than 10 years. Many Mabs are already in clinical trials. The production capacity of MAbs was 4,000 kg in 2005 and the capacity is currently (2009) estimated to 10,000 kg. Most of this capacity is provided by stirred tanks having a capacity of around 1 mio. liters. This capacity may not even be sufficient for the production of the few, so far clinically validated therapeutic antibodies.
A projected demand of an expected capacity of 50,000 kg/year by 2020 will require at least 5 times the present, conventional stainless steel stirred tank capacity. It is clear that a World wide bio-tech industry will need to invest heavily in more production capacity in order to increase the current production capacity.
There is clearly a need for novel and innovative bio-reactor devices as well as for alternative methods for the production of bio-active compounds using such bio-reactor devices.
The market for bio-reactors can be divided into the following groups:                Bench-top equipment market, 1-10 liter size        Pilot-scale equipment market, 10-100 liter size        Small-production equipment market, 100-1000 liter        Large-production equipment market, >1000 liter        
Current fermenter technologies exhibit a relatively unattractive surface-to-volume ratios (stirred tanks≈1 m2/40 liters and hollow fibres≈1 m2/10 liters). Scaling up bio-reactors for use in the production of adherent micro organisms today relies exclusively on the possibility of increasing the surface area that is available for the adhesion of the micro organisms. This can be done e.g. by increase the size of the reactors. Classical micro organism culture lab ware ranges from 1 to 500 cm2 per stationary flask. The surface area to volume ratio is the range of from 1,000 to 2,000 cm2/liter for rotating flasks. Rotating wall vessel systems on the market would at best reach a ratio of 7.5 m2 in a 2.5 m3 volume.
Multi-plate systems, like OptiCell from Nunc, require large, thermo-controlled volumes to be efficient when used with a high surface area. To face these limitations, micro-carrier approaches, e.g. Cytodex from GE Healthcare) have been developed and such micro-carriers can bring the surface area up to approx. 9 m2/liter for stirred suspension cultures where micro organisms are sensitive to shear forces due to stirring—thus covering only partially the available surface area with micro organisms.
Besides the limitation of the surface area available for micro organisms anchoring, additional problems exist, including cultivation difficulties, undesirable oxygen gradients, tension control, pH control and a general lack of homogeneity of nutrient delivery throughout the bio-reactor.
Micro organism expression levels depend upon the type of molecule to be produced, which range from approx. 5 mg/liter/day for laboratory clones to up to 1 g/liter/day under ideal industrial condition. Based on such production rates, the expected production achievable with a 20 liter continuous, wall-forced-flow bio-reactor is a daily, steady state production of up to 2 grams of bioactive compound per day. Due to expected benefits related to bio-reactor geometry, surface properties, flow control and distribution of nutrients, these production rates might be further improved. By comparison, a classical batch bio-reactor based on micro carriers and duration of 8-10 days may at best produce 10 mg per liter. Accordingly, a 20 liter bio-reactor would produce at the most 200 mg bioactive compound per batch.
Conventional Methods and System Designs
Bio-reactor design is a complex engineering task. Under optimum conditions, the micro organisms must be able to perform their desired function with a 100 percent rate of success. The bio-reactor's environmental conditions, like gas content (i.e., air, oxygen, nitrogen and carbon dioxide), flow rates, temperature, pH and agitation speed/circulation rate need to be closely monitored and controlled. In a traditional, batch bio-reactor the nutrient volume is constant—it is not exchanged and it is stirred throughout the duration of the fermentation.
In a Continuous Flow Stirred Tank bio-reactor (CSTR or chemostat), the continuous flow of fresh medium is fed into the bio-reactor at a constant rate, and medium is mixed with micro organisms and an undesirable mix of cells and product leaves the bio-reactor at the same rate. A fixed bio-reactor volume is maintained and ideally, the effluent stream should have the same composition as the bio-reactor contents. The culture is fed with fresh medium containing one and sometimes two growth-limiting nutrients, including e.g. glucose. The concentration of the micro organisms in the bio-reactor is controlled by the concentration of the growth limiting nutrient level. A steady state micro organism concentration is reached when the density of the micro organisms and the nutrient concentration are constant.
A bio-reactor is a suitable device for growing both anchorage dependent and suspended micro organism. Ideally, any micro organism culture bio-reactor must maintain a sterile culture of micro organisms under medium conditions which maximize micro organism growth and productivity. In the aerobic process, optimal oxygen transfer and access is perhaps the most difficult task to accomplish. Oxygen is poorly soluble in water at atmospheric pressure, even less in fermentation broths, usually helped by agitation, which is also needed to mix nutrients and to keep the fermentation homogeneous. There are, however, limits to the speed of agitation in stirred tank bio-reactors, due both to high power consumption and damages to the micro organism.
In bio-reactors where the goal is to cultivate micro organisms or tissue cells for experimental or therapeutic purposes, the design is often significantly different from industrial bio-reactors. Many micro organisms and tissues, especially mammalian ones, must have a surface or other structural support in order to grow, and agitated environments are often destructive to these micro organism types and tissues. Higher organisms also need more a complex growth medium composition. The most crucial step in the design of monolithic reactors is the proper distribution of nutrient fluids over the reactor cross section.
Flasks for laboratory use are made from either glass or polycarbonate and very commonly they are used only for small scale productions. The maximum surface available in a single flask is 500 cm2 as offered from Thermo Fishers Danish department NuncBrand in the TripleFlask model. If stacked in a one meter high setup the flasks can offer a surface area of 0.6 m2/20 liters volume into a manually handled process.
The German company Sartorious market CELLine cultivation flasks with a built-in micro organism separation method offering nutrient re-circulation and easier product harvesting. Flask surface areas range from 350 cm2 to 1000 cm2 for anchored micro organisms. Also available is a cassette based, stackable system for small, industrial micro organism cultivation on porous polymeric membranes. Each cassette offers 100 cm2 area and 20 cassettes fit into a ready made cassette support system offering in total adhesion area of 2,000 cm2. Each cassette has its own two resealing access ports which provide a closed, growth environment with a sterile fluid path. It is purely intended for anchorage-dependent cells.
Industrial vessel fermenters from Novaferm in Sweden or NNE (Novo Nordisk Engineering) in Denmark offer cultivation systems based on micro organisms suspended in the nutrient. The contact between nutrients and the micro organism are supported by mechanical stirrers and this is far from optimal.
Packed bed bio-reactors with small polyester fibre bodies, like the FibraCell product from New Brunswick Scientific in the United States, features an axial flow path along the porous bodies typically enclosed in a glass vessel. The FibraCell product is originally developed in the early 1990ties by Mr. Avinoam Kadouri from Yeda and Weizmann Institute of Science, Israel, but already in the 1980ties had exceptionally high yields, typically 108 micro organisms/cm3 of bed volume depending on selected micro organisms, been reported. The small bodies are less than 6 millimeter in diameter and the polyester fibre diameter is approx. 6 μm, bulk gravity 1 g/cm3, and the available growth surface areas >1200 cm2/g. The CelliGen 310 type of bio-reactor, also from New Brunswick Scientific—when packed with 500 gram Fibra Cell mini bodies (and in general for small scale industrial application with 14 liters volume)—offer 500 gram×1200 cm2=25 m2 surface area equivalent to 5 m2/liter. The flow path is exclusively axial in the columns. Greater height is generally the solution when seeking to add more packing volume and this demands a greater volumetric flow in the axial direction and this in turn increases the risk of cell damage along the packed bed. Furthermore, the longer the bed the higher is the risk of experiencing an increased lack of nutrients and CO2 content in the opposite end of the fluid inlet. As the nutrient flow now also becomes the exhaust fluid, the flow in the axial direction has a maximal limit. Cell densities of up to 1×108 and expression levels of approx. 0.5 g/mL have been reported from various sources. Artellis from Belgium has recently started to promote such a technology with small polyester fibre bodies packed in stackable envelopes.
Packed bed bio-reactors with porous micro beads, such as micro spheres provided by GE Healthcare or others, are in general very expensive. The beads are typically filled with macro pores and typical have a diameter of from 0.01 millimeter to up to 5 millimeter. They are manufactured from e.g. cellulose, polyethylene glass, gelatine, silicone rubber, silica, etc. Cell densities of up to 1×108 have been reported with the use of micro beards.
Membrane bio-reactors (MBR) are bio-reactors having a cross flow filtration unit which enables continuous processes with total micro organism retention within the reactor to be realized. Provided that high dilution rates can be applied and that inhibition processes are avoided, high biomass concentrations can be reached, thereby improving the volumetric productivity. These membrane bio-reactors have been successfully applied to various microbial bio conversions, such as alcohol fermentation, solvents, organic acid production, starters, and wastewater treatment. On the basis of the biological reaction characteristics and bibliographic results available, there are several bottlenecks associated with this methodology.
Hollow fibre bio-reactors systems use polymeric round hollow fibres bundles into a circular body with all the fibres passing axially through the core. The hollow fibres system can be used for anchorage dependent and suspension micro organisms. Typically the hollow fibre on the inside of the tube is provided with a membrane giving selective passage of molecules depending on their size. In most cases, an ultra filtration type of membrane is used. The molecular cut-off of the membrane differs according to applications, ranging from a few thousand to a hundred thousand Daltons. The ultra filtration membrane prevents free diffusion of secreted product molecules from passing through the membrane and allows them to accumulate in the extra capillary space to a high concentration. The culture media is pumped usually through the fibre lumen, and micro organism grow in the extra capillary space, or the shell side. Supply of low-molecular weight nutrient to the micro organisms and the removal of waste product occur by diffusive transport across the membrane between the lumen and the shell spaces. Although the use of micro filtration hollow fibre membranes for micro organism culture is infrequent, it does find application in various research uses for studying metabolism and for the cultivation of anchorage-dependent or highly aggregated micro organism for which a convective flow of medium through the extra capillary space to bathe micro organism in medium is desired. Scaling-up of a hollow fibre system eventually is limited by the ability to extend the axial length of the fibre without incoming oxygen transfer limitation, creating gradients. Also scaling up the cartridge diameter eventually runs into flow distribution problems among thousands of fibres. The relatively high surface area to volume ratio that hollow fibre bio-reactors provide (100 cm2/mL or more) and the high flux rates of fibres from FiberCell Systems Inc. can allow micro organism to grow at 100 times the density found in flask culture (10*8 vs. 10*6/mL in flask) and is in fact one of the only cell culture methods that permits micro organism cultures at densities that rival those found in vivo.
Honeycomb ceramic bio-reactor systems are typically in the form of a cylinder made of a porous ceramics with square channels passing through the ceramic cylindrical body in the longitudinal direction. Micro organisms are inoculated into the channels and either adheres to the surface or become entrapped into the pores of the ceramic body walls. Medium is passed through the channels to provide nutrient and to remove the metabolites. In the ceramic system, the side on which the micro organisms is adhered is exposed to a slow stream of permeate. The ceramic bio-reactor, to some extent, can be considered a variant of the hollow fibre system described herein above. As in the hollow fibre system, ceramic reactors are supported by medium perfusion loops. Micro organism culture medium is pumped through the longitudinal channels in the ceramic core from a medium reservoir in a circulating loop configuration. Fresh medium is fed into the system, and harvested bioactive compounds are removed to the medium reservoir. Unlike the hollow fibre system, there is no membrane separating the micro organisms and the bulk medium. Products are secreted directly into the bulk medium. Essentially, the ceramic bio-reactor (like the OptiCell) can be used to conveniently replace a large number of roller bottles. As in the hollow fibre systems, oxygen concentration gradient develops along the axial direction and poses a limitation to the length and/or the physical size of the ceramic reactor.
A laminar flow along a channel is often not sufficient to establish a contact between nutrients and a micro organism. Turbulence is not created along the channels having relatively smooth surfaces and the flow will generally be lower at the edges compared to the flow at the flat wall between the edges. Attempts to increase channel density only serves to increase this problem. The air-lift type of membrane bio-reactors do cause some turbulence, but the bubble sizes prevents the oxygen containing bubbles from coming into contact with the micro organisms in the corners. Also, it has been reported that the bubbles may damage the cells. As there is no force to create an interaction between the many channels the nutrients flow along, the channels are not fed equally. More channels in parallel will not affect the flow, the mass and the nutrient transfer to the cells. The liquid distribution results in the provision of heterogeneous sub-environments and a non-uniform protein production. This in turn seriously limits the applicability and the commercial potential for using ceramic monoliths having square cells as bio-reactor supports for the cultivation of micro organisms in large scale fermentations.
Plastic bags, disposable bags are becoming the biggest seller today below 1,000 liter bio-reactor volume. Bags with media inside only need a mechanism inside the bag in order to constantly mix the nutrient with the suspended micro organism. Many different techniques are on the market based on rotating devices with direct mechanical or magnetic force transfer or waving actions. However, the mixer typically adds shear forces to the media and this may potentially damage the micro organisms. Micro carriers could also be used with the bag operation in a perfusion system without a mixer relying on external, positive displacement pumps.
Wave-bag is a single-use system which rock, wave a volume of nutrient in a flat plastic bag on a tray which is kept in simple motion by an external mechanical force. Typical max cell densities are in the range of from 1-2×107/mL when operating in perfusion mode.
U.S. Pat. No. 4,948,728 (G. Stephanopoules) discloses a porous, ceramic material with a plurality of flow passages and a biofilm in contact with an inner wall in a channel and a gas permeable membrane covering an outer wall. A separate oxygen flow along the outer wall permeates the membrane and ceramic housing to reach the biomaterial. Nutrients flow along the inner wall in direct contact with the biofilm. The basic idea behind the invention is to apply oxygen on one side and nutrient flow on the other side of a porous wall. The monoliths, as supplied in the late 1980ties from Corning, are all flow through monoliths originally intended for automobile catalyst support and described in the patent as having a cell density of either 200 cells/in2 (cpsi) (column 10) or 300 (column 5) cells/in2 (cpsi). The Cordierite ceramic monoliths used are characterised as having a pore size of less than 15 μm. The expression “race track” monoliths which is used in the specification indicates that the catalyst carriers originate from the automobile industry and have very thin walls—typically less than 0.5 millimeter. A maximum cell density—with every second channel blocked and designed for DPF (diesel particulate filter) use—would be 90 cells per square inch (cpsi). The use of a parallel wall DPF is not described in the US patent or any associated literature. In the '728 patent, FIG. 1 illustrates a flow through the monolith. FIG. 4 shows the oxygen appearance on one side of the porous wall and the nutrient appearance on the other side of the porous wall. FIG. 5 illustrates that the penetration of oxygen into the porous ceramic wall is not complete, but only partial. Accordingly, there is no forced, hydraulic nutrient or oxygen flow through the wall through which e.g. glucose and oxygen is supposed to flow. This means that the predominant amount of bio film does not receive oxygen and this in turn results in a reduced production capacity. FIG. 5 also shows that the bio film penetration is limited by the hydrophobic membrane and this results in reducing the total amount of micro organisms inside the matrix. The '728 patent does not disclose e.g. how the channel flow should be adapted and/or controlled, if the channels should be blocked for flow control, or how the separation of inlet/outlet channels should be arranged.
Later documentation from G. Stephanopoules shows that controlling the two different fluid flows is not solved via channel blocking, but can be solved via the introduction of multiple, porous ultra small diameter silicon rubber hoses one of which is placed in each channel in order to supply the oxygen. However, this is very impractical and more or less impossible on a ceramic body with 200 or 300 cells/in2, where each channel width is only 1 millimeter or less.
Also, the '728 patent does not disclose any membrane separating the inlet from the outlet. Such a membrane would be required in order to prevent cells from passing through the bio-reactor and be expelled in the flow after the reactor. Also, a high pressure drop membrane playing an important factor in distribution of oxygen and nutrients is not mentioned at all.
Reference is also made to the article by M. A. Applegate and G. Stephanopoulos, “Development of a Single-Pass Ceramic Matrix Bio-reactor for Large-Scale Mammalian Cell Culture,” Biotech methodology and Bio engineering, volume 40, 1992, pp. 1,056-1,068. The use of porous hoses by Applegate and Stephanopoulos in each channel in order to supply oxygen (separate oxygenation system) is also described in the article “Review of Nonconventional Bio-reactor Technology” by C. E. Turick published 1993 by U.S. Department of Energy. On page 13, the cited equation refers to open channel ceramic bodies. FIG. 11 on page 12 illustrates one porous silicon tube in each channel in order to supply air/oxygen. It is obvious that channel closing has not been part of the patented invention. One other drawback of using Cordierite is the well known fact that a considerable amount of the pores as described by the porosity figure are so called closed pores. In other words there is no access to as much as 25% of the pores as they are not interconnected to other pores. The product reached a commercial status and became known as the OptiCell product in the industry.
U.S. Pat. No. 4,514,499 (Corning, Inc) describes a monolithic support for cell growth with the same general problems as describes above for U.S. Pat. No. 4,948,728.
U.S. Pat. No. 4,937,196 (New Brunswick Scientific) introduces the terms “membrane” and “micro porous membrane”. A micro porous membrane does NOT allow the cells to pass the membrane. It is disclosed that “the dimension of the system is such that every cell is within 200 μm, preferably within 100 μm, of the oxygen source, i.e., the surface of an oxygen transport membrane”. The oxygen supply is separated from the nutrient supply (which is void of oxygen) as the two individual feed spacers are separated by the membrane containing the cells. The laminated set-up disclosed in the '196 patent imposes the physical limit that the membrane containing the cells can have a maximum thickness of about 200 μm—as the oxygen supplying spacer is present only on one side of the cell supporting membrane.
Furthermore, it is described that the membranes are positive and do not allow the cells to pass the membrane. FIG. 9 illustrates the fluid path along the spacers from one end to the other, but no dual path feed spacer system capable of supporting such a teaching is disclosed. Nowhere does the '196 patent disclose how the individual feed spacers are anchored to the central tube in the spiral filter. Also, the legend to FIG. 10 indicate that a significant and undesirable pressure gradient is present, but the legend does not offer any suggestion as to how to avoid this.
U.S. Pat. No. 5,501,971 (New Brunswick Scientific) describes the Celligen system in the form of a packed bed of FibraCell polyester fibre based, flat carriers. The system currently on the market from New Brunswick Scientific demonstrates that cells adhere well to a polyester fibre, but the Celligen system is heavily limited by the thickness of the bed. As an increased bed thickness introduces or increases undesirable gradients in the bio-reactor, the bio-reactor device is not scalable and can be used for fermentations on a bench-top scale only.
U.S. Pat. No. 5,266,476 (Yeda) discloses a matrix of a thickness of from 50 to 500 μm and a surface area of 1 millimeter2. The matrix is based on a polyester fibre and is in the form of a very small carrier know in the industry under the trade name FibraCell (supplied by both NBS and Bibby Sterilin Ltd, UK). FibraCell is a mini body approx. 6 millimeter in diameter and 0.6 millimeter thick. FibraCell has been used for various applications, among others by NBS (New Brunswick Scientific, USA) for manufacturing the Celligen product in which Fibra Cell bodies are randomly and efficiently packed in volumes of up to 1 liter. Larger reactor volumes are not possible due to gradient problems.
U.S. Pat. No. 4,546,083 (Stoll Corp) discloses a cylindrical device with fibres positioned around a tubular inlet—a spindle. The volume for the fibre matrix between the spindle and the cartridge inner wall are filled with fibres only.
U.S. Pat. No. 5,543,047 (Pall, Inc) describes a special pleating method improving among others also the potential area/volume ration and as described for filtration purposes only.
U.S. Pat. No. 5,563,069 (Ohio State University) is concerned with a packed bed structure based on cotton cloth in sheet shape.
WO2007/142664 (AMProtein Corp) discloses a method for increasing dissolved oxygen in a culture medium for a semi suspension culture of mammalian cells in a vessel. The vessel concept exploits constant height and varies the diameter in order to vary the fermenter volume in order to handle the gradient issue.
WO2007/039600 (Artelis) discloses a complicated flow pattern vessel device, which has an integrated mixer operating as circulation pump. The device is not suitable for up-scaling due to an undesirable increase in nutrient and/or oxygen gradients. Media re-circulation is performed by a mixer device internally in the vessel and this feature also seriously limits the prospects for up-scaling. Only perfusion mode operation is described. Gradients occurring in the central inlet zone results in the generation of an un-even cell density in the modules. The invention is based on packed micro porous micro carriers present inside modules, envelopes for cell support and/or empty modules suitable for the suspension of cells. Each radial fluid inlet between two envelopes supply media to only one of two envelopes. As described, most of the cells are not circulated, but those which are will be subjected to strong shear forces generated by the mixer impeller. Such cells are at risk of being seriously damaged. The described mixer physics exploit very limited pressure difference capabilities, both limiting the scale-up of the concept and increase shear forces in the fluid significantly.
U.S. Pat. No. 4,789,634 (KG Biologische Laboratorien) discloses a circular devise used for containing polymer microspheres, such as beads. The beads are located in round, stackable sieve boxes to ensure both a radial and an axial flow path.
The journal “BioProcess International” published in June of 2009 carries an article wherein AMprotein (China) states that they use large, randomly oriented, loosely packed cellulose fibre bodies, or Rasching alike soft elements, as “macro”-carriers for bio-reactor fermentations. It appears that the “Current” bio-reactor packed bed height is constant at approx. 150 millimeter and that the volume is modified by using 3 different diameters according to a reactor size ranging from 5 liters over 50 liters to 150 liters, thereby avoiding the problem of how to solve the problem of gradient formation.
MembroFerm is a product which was marketed by MBR Bio Reactor AG (Switzerland) in the early 1990ties. The product is in the form of a flat membrane bio-reactor separated by a thin film fluorocarbon matrix having a thickness of 0.6 millimeter.
None of the above-cited prior art references have solved the gradient problems associated with “packed bed” bio-reactor fermentations. Also, none of the above-cited prior art references have solved the problem of how to effectively scale a fermentation from a bench-top level to industrial production scale without the concomitant generation of undesirable gradients in a bio-reactor.